Spot-Trap

Immunoprecipitation of Spot-tagged protein. Input (I), non-bound (FT) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining and Western blotting. Western blot indicates high effectiveness of pull-down.

Spot-Trap is a very low background affinity reagent. See benchmark of Spot-Trap Agarose and Magnetic Agarose with other suppliers’ capture tools. Immunoprecipitations were conducted from HEK293T cell lysates without Spot-Tag present and normalized for equal binding capacity. The IPs were done according to respective manufacturers’ protocols. It is apparent that Spot-Trap does not only lack heavy and light chain antibody contaminations but also unspecific binding. This makes Spot-Trap a superior affinity capture tool for single band purification of Spot-tagged proteins.

Elution of affinty purified, Spot-tagged protein. Input (I), non-bound (FT) and eluted (E1, E2 and E3) fractions were separated by SDS-PAGE followed by Coomassie blue staining and Western blotting. Elution with Spot-peptide. Western blot indicates effective elution.

Description

The ChromoTek Spot-Trap^® is an affinity resin for immunoprecipitation and affinity purification of Spot-tagged proteins.
It consists of an anti-Spot-Tag Nanobody/ V_HH, which is conjugated to beads.

Applications
Immunoprecipitation (IP)/ Co-IP
IP/ Co-IP for mass spec (MS) analysis
Affinity purification -> see Spot-Cap

Specificity
Spot-Tag is an inert 12 amino acid peptide tag with the sequence PDRVRAVSHWSS and can be cloned to N- and C-terminus.
Please find more information about the Spot-Tag here.

Formats/matrices
Spot-Trap Agarose
Spot-Trap Magnetic Agarose
Spot VHH, recombinant binding protein

Synonyms
anti-Spot V_HH, Spot binding protein, Spot Nanobody, or anti-Spot single domain antibody fragment (sdAb)

Benefit from Spot-Trap for Immunoprecipitation (IP)

Reduced background
High binding affinity, low dissociation constant KD of 6 nM for Spot fusion proteins
Harsh washing conditions
High reproducibility
Short incubation time of 30-60 minutes
Extraordinary low background compared to other affinity resins

Format/matrices & properties

We offer the Spot VHH coupled to agarose beads and magnetic agarose beads, or uncoupled as purified protein.

	Spot-Trap agarose	Spot-Trap magnetic agarose
matrix	agarose	magnetic agarose
color	white	black
particle size	90 µm	40 µm
binding capacity	1.4 µg/µL	1.4 µg/µ
magnetic	no	yes
may be centrifuged up to	2,500 x g	800 x g

Regeneration of Spot-Trap beads is possible. It is recommended, however, to re-use beads for the repeated purification of the same protein only to avoid cross-contamination.

Specificity

Spot-Trap binds to the sequence motif PDRVRAVSHWSS.

Spot-Trap is a very low background affinity reagent. See benchmark of Spot-Trap Agarose and Magnetic Agarose with other suppliers’ capture tools. Immunoprecipitations were conducted from HEK293T cell lysates without Spot-Tag present and normalized for equal binding capacity. The IPs were done according to respective manufacturers’ protocols. It is apparent that Spot-Trap does not only lack heavy and light chain antibody contaminations but also unspecific binding. This makes Spot-Trap a superior affinity capture tool for single band purification of Spot-tagged proteins.

About Spot

ChromoTek’s proprietary and novel Spot-System is the first peptide-tag specific Nanobody for universal capture & detection applications. It comprises Spot-Tag^®, an inert 12 amino acid peptide-tag (PDRVRAVSHWSS), which has been affinity optimized, and the universal, rugged Spot-Nanobody that specifically binds to Spot-tagged proteins with high affinity.
Spot-Tag peptide is derived from the protein beta-catenin: its sequence has been optimized for higher affinity binding to the Spot-Tag Nanobody. For further information see here.